Journal Article

Elucidation of the Fe(iv)=O intermediate in the catalytic cycle of the halogenase SyrB2

Zhao, Jiyong, Wong, Shaun D., Liu, Lei V., Srnec, Martin, Seto, Makoto, Kwak, Yeonju, Yoda, Yoshitaka, Bollinger, J. Martin, Park, Kiyoung, Matthews, Megan L., Bell III, Caleb B., Solomon, Edward I., Kitao, Shinji, Alp, E. Ercan, Krebs, Carsten

2013

Overview

Synchrotron-based nuclear resonance vibrational spectroscopy is used to characterize the reactive Fe( iv )=O intermediate of the halogenase SyrB2; the substrate directs the orientation of this intermediate, presenting specific frontier molecular orbitals that can activate the selective halogenation. Structure of a halogenase SyrB2 reactive intermediate A key reactive intermediate in the oxidative reactions catalysed by mononuclear non-haem iron (NHFe) enzymes is the high-spin species S = 2 Fe( IV ) = O. In this manuscript the authors use synchrotron-based nuclear resonance vibrational spectroscopy (NRVS), a sensitive method that defines the dependence of the vibrational modes of Fe on the nature of the Fe( IV ) = O active site, to determine the structure of the reactive intermediate of the halogenase SyrB2 from the plant pathogen Pseudomonas syringae . This intermediate reacts via an initial hydrogen-atom abstraction step, with its subsequent halogenation (native) or hydroxylation (non-native) rebound reactivity being dependent on the substrate. The substrate directs the orientation of the oxo intermediate, presenting specific frontier molecular orbitals that can activate the selective halogenation versus hydroxylation reactivity. Mononuclear non-haem iron (NHFe) enzymes catalyse a broad range of oxidative reactions, including halogenation, hydroxylation, ring closure, desaturation and aromatic ring cleavage reactions. They are involved in a number of biological processes, including phenylalanine metabolism, the production of neurotransmitters, the hypoxic response and the biosynthesis of secondary metabolites 1 , 2 , 3 . The reactive intermediate in the catalytic cycles of these enzymes is a high-spin S = 2 Fe( iv )=O species, which has been trapped for a number of NHFe enzymes 4 , 5 , 6 , 7 , 8 , including the halogenase SyrB2 (syringomycin biosynthesis enzyme 2). Computational studies aimed at understanding the reactivity of this Fe( iv )=O intermediate 9 , 10 , 11 , 12 , 13 are limited in applicability owing to the paucity of experimental knowledge about its geometric and electronic structure. Synchrotron-based nuclear resonance vibrational spectroscopy (NRVS) is a sensitive and effective method that defines the dependence of the vibrational modes involving Fe on the nature of the Fe( iv )=O active site 14 , 15 , 16 . Here we present NRVS structural characterization of the reactive Fe( iv )=O intermediate of a NHFe enzyme, namely the halogenase SyrB2 from the bacterium Pseudomonas syringae pv. syringae . This intermediate reacts via an initial hydrogen-atom abstraction step, performing subsequent halogenation of the native substrate or hydroxylation of non-native substrates 17 . A correlation of the experimental NRVS data to electronic structure calculations indicates that the substrate directs the orientation of the Fe( iv )=O intermediate, presenting specific frontier molecular orbitals that can activate either selective halogenation or hydroxylation.

• Publisher: Nature Publishing Group UK,Nature Publishing Group
• Subject: 631/45/49/1141 / 639/638/440/527 / Analysis / Biocatalysis / Biosynthesis / Crystal structure / Enzymes / Halogenation / Humanities and Social Sciences / Hydroxylation / Hypoxia / Iron - chemistry / Iron in the body / letter / Ligands / Metabolites / Methods / multidisciplinary / Oxidoreductases - chemistry / Oxidoreductases - metabolism / Physiological aspects / Pseudomonas syringae - enzymology / Science / Secondary metabolites / Spectrum analysis